Bottom Line:
By RNA-seq analysis, only 212 genes (0.8% of detected genes) are found significantly differentially expressed between 2x and 4x leaves.However, the effect of tetraploidization on transcriptome is limited.Compared to diploid, higher expression level of stress related genes and higher content of stress related metabolites in doubled diploid could be beneficial for its stress tolerance.

Background: Polyploidy has often been considered to confer plants a better adaptation to environmental stresses. Tetraploid citrus rootstocks are expected to have stronger stress tolerance than diploid. Plenty of doubled diploid citrus plants were exploited from diploid species for citrus rootstock improvement. However, limited metabolic and molecular information related to tetraploidization is currently available at a systemic biological level. This study aimed to evaluate the occurrence and extent of metabolic and transcriptional changes induced by tetraploidization in Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka), which is a special citrus germplasm native to China and widely used as an iron deficiency tolerant citrus rootstock.

Results: Doubled diploid Ziyang xiangcheng has typical morphological and anatomical features such as shorter plant height, larger and thicker leaves, bigger stomata and lower stomatal density, compared to its diploid parent. GC-MS (Gas chromatography coupled to mass spectrometry) analysis revealed that tetraploidization has an activation effect on the accumulation of primary metabolites in leaves; many stress-related metabolites such as sucrose, proline and γ-aminobutyric acid (GABA) was remarkably up-regulated in doubled diploid. However, LC-QTOF-MS (Liquid chromatography quadrupole time-of-flight mass spectrometry) analysis demonstrated that tetraploidization has an inhibition effect on the accumulation of secondary metabolites in leaves; all the 33 flavones were down-regulated while all the 6 flavanones were up-regulated in 4x. By RNA-seq analysis, only 212 genes (0.8% of detected genes) are found significantly differentially expressed between 2x and 4x leaves. Notably, those genes were highly related to stress-response functions, including responses to salt stress, water and abscisic acid. Interestingly, the transcriptional divergence could not explain the metabolic changes, probably due to post-transcriptional regulation.

Conclusion: Taken together, tetraploidization induced considerable changes in leaf primary and secondary metabolite accumulation in Ziyang xiangcheng. However, the effect of tetraploidization on transcriptome is limited. Compared to diploid, higher expression level of stress related genes and higher content of stress related metabolites in doubled diploid could be beneficial for its stress tolerance.

Fig2: Principal component analysis of GC-MS metabolite profiling data from 4× and 2× leaves. First two components could explain 75.8% of the metabolite variance. Component 1 explained 58.6% of the variance and component 2 explained 17.2%.

Mentions:
Principal component analysis (PCA) served as an unsupervised statistical method to study the differences of the major metabolites of 4× and 2× (Figure 2). Parameters of the PCA model based on the primary metabolic data were: two principle components were calculated by cross validation, 58.6% of variables can be explained by first component and 17.2% of variables can be explained by the second component. A clear separation trend could be observed in the score plot (Figure 2), implying that extensive changes in the major metabolites were induced by tetraploidization.Figure 2

Fig2: Principal component analysis of GC-MS metabolite profiling data from 4× and 2× leaves. First two components could explain 75.8% of the metabolite variance. Component 1 explained 58.6% of the variance and component 2 explained 17.2%.

Mentions:
Principal component analysis (PCA) served as an unsupervised statistical method to study the differences of the major metabolites of 4× and 2× (Figure 2). Parameters of the PCA model based on the primary metabolic data were: two principle components were calculated by cross validation, 58.6% of variables can be explained by first component and 17.2% of variables can be explained by the second component. A clear separation trend could be observed in the score plot (Figure 2), implying that extensive changes in the major metabolites were induced by tetraploidization.Figure 2

Bottom Line:
By RNA-seq analysis, only 212 genes (0.8% of detected genes) are found significantly differentially expressed between 2x and 4x leaves.However, the effect of tetraploidization on transcriptome is limited.Compared to diploid, higher expression level of stress related genes and higher content of stress related metabolites in doubled diploid could be beneficial for its stress tolerance.

Background: Polyploidy has often been considered to confer plants a better adaptation to environmental stresses. Tetraploid citrus rootstocks are expected to have stronger stress tolerance than diploid. Plenty of doubled diploid citrus plants were exploited from diploid species for citrus rootstock improvement. However, limited metabolic and molecular information related to tetraploidization is currently available at a systemic biological level. This study aimed to evaluate the occurrence and extent of metabolic and transcriptional changes induced by tetraploidization in Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka), which is a special citrus germplasm native to China and widely used as an iron deficiency tolerant citrus rootstock.

Results: Doubled diploid Ziyang xiangcheng has typical morphological and anatomical features such as shorter plant height, larger and thicker leaves, bigger stomata and lower stomatal density, compared to its diploid parent. GC-MS (Gas chromatography coupled to mass spectrometry) analysis revealed that tetraploidization has an activation effect on the accumulation of primary metabolites in leaves; many stress-related metabolites such as sucrose, proline and γ-aminobutyric acid (GABA) was remarkably up-regulated in doubled diploid. However, LC-QTOF-MS (Liquid chromatography quadrupole time-of-flight mass spectrometry) analysis demonstrated that tetraploidization has an inhibition effect on the accumulation of secondary metabolites in leaves; all the 33 flavones were down-regulated while all the 6 flavanones were up-regulated in 4x. By RNA-seq analysis, only 212 genes (0.8% of detected genes) are found significantly differentially expressed between 2x and 4x leaves. Notably, those genes were highly related to stress-response functions, including responses to salt stress, water and abscisic acid. Interestingly, the transcriptional divergence could not explain the metabolic changes, probably due to post-transcriptional regulation.

Conclusion: Taken together, tetraploidization induced considerable changes in leaf primary and secondary metabolite accumulation in Ziyang xiangcheng. However, the effect of tetraploidization on transcriptome is limited. Compared to diploid, higher expression level of stress related genes and higher content of stress related metabolites in doubled diploid could be beneficial for its stress tolerance.